S-nitrosylation contributes to the control of cell death, regulating several apoptotic pathways including p2 lras, NF-kappaB, and the cysteine proteases (i.e., caspases). We have shown that perturbation in the level of intracellular nitrosothiol (SNO) leads to apoptosis ofmacrophages, lymphocytes, and bronchial epithelium. This finding is significant as apoptosis plays a role in the resolution of airway inflammation and airway remodeling seen in asthma. Interestingly, we have determined that the level of airway SNO decreases in asthma. The decrease in airway SNO occurs despite higher levels of exhaled NO from the lungs of asthmatics. This paradox is best explained by enhanced SNO degradation in the asthmatic airway. Importantly, we have identified an enzyme, GSNO reductase, that is the primary regulator of intracellular SNO. Preliminary studies in a GSNO reductase knockout mouse indicate hyporesponsiveness to airway provocation, which is consistent with our hypothesis that SNO metabolism is involved in the pathogenesis of asthma. Given our recent discoveries in SNO metabolism as it relates to apoptosis and the asthma phenotype, we have formulated the following specific aims: 1. Characterize the changes in airway SNO and SNO metabolism in asthma. 2. Determine if modulation in airway SNO in asthma results in a dysregulation of airway cell apoptosis. 3. Measure the asthmatic response in mice with targeted deletion of GSNO reductase. 4. Search for mutations in the GSNO reductase gene in an established human asthma cohort. We will quantify the changes in airway SNO and SNO metabolism in human subjects with asthma (atopic and non-atopic patients), in two murine models of asthma (innate and allergic asthma models), and in bronchial epithelial cells in vitro (unstimulated and cytokine-stimulated). The alteration in airway SNO and SNO metabolism will be correlated to physiologic (i.e., airway resistance) and histopathologic (i.e., number and type of airway inflammatory cells) parameters of asthma in the murine models and human asthmatics. In addition, we will determine if changes in airway SNO metabolism relate to a modulation in apoptosis of the airway epithelium (both in vivo and in vitro) or airway inflammatory cells. The response of wild type versus GSNO reductase knockout mice will be compared using the two, different murine asthma models. Finally, we will utilize banked DNA from an established human asthma cohort (GAIN study) as well as an African-American asthma cohort (Project 2) to search for mutations in the coding sequences of the human GSNO reductase gene. Upon completion of these studies, significant insight into the pathogenic role of NO in asthma will be attained with the possibility that novel therapeutic strategies can be developed.